Peptides from pseudomonas syringae possessing broad-spectrum antibiotic activity

ABSTRACT

Peptide antimycotics, termed pseudomycins, display broad spectrum antibiotic activity, and in particular are highly effective, non-toxic antibiotics against fungal pathogens of human and animal disease. The peptide antimycotics (pseudomycins) may be used in the treatment of the fungal pathogen Candida albicans. Also disclosed is a method of purification and isolation, including characterization, of the pseudomycins.

The United States Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms of GrantNos. DMB 8607347 and DIR 18937 awarded by the National ScienceFoundation.

This application is a continuation-in-part application of Ser. No.07/982,687, filed Nov. 30, 1992, now abandoned.

TECHNICAL FIELD

The present invention relates to peptide antimycotics, termedpseudomycins, which display wide spectrum antibiotic activity, and inparticular are highly effective, non-toxic antibiotics against fungalpathogens of human and animal disease. In a preferred embodiment, thepeptide antimycotics (pseudomycins) according to the present inventionare particularly useful in the treatment of the fungal pathogen Candidaalbicans. The peptide antimycotics (pseudomycins) are also effective inthe treatment of plant disease and may additionally be used asherbicides.

The present invention also relates to the method of purification andisolation, including characterization, of the peptide antimycotics(pseudomycins).

BACKGROUND OF THE INVENTION

Fungi adversely affect the health and well being of mankind in numerousways. The most direct of these ways include a variety of diseaseprocesses affecting humans, such as opportunistic infections inimmunologically compromised patients, such as patients afflicted withAIDS. The most common treatment currently available for these fungalinfections is administration of the antibiotic amphotericin B. However,this compound is highly toxic and doses must be kept to a minimum,especially in critically ill patients. Doses are thus often in amountsinsufficient to cure the disease or even halt the infection.

Other harmful effects of fungi include the adverse economic and socialeffects of plant disease. Purified natural herbicides have at least twoadvantages; 1) as weed control agents they have longer shelf life, widerrange of storage conditions, a broader environmental window forapplication, less storage space, and a greater ease of application thanwith living organisms and 2) new microbial phytotoxins may be useful inexpanding the number of sites of action of herbicides, in that there islittle overlap between the known sites of action of commercialherbicides and of microbially-produced phytotoxins.

Pseudomonas syringae represents a wide range of plant-associatedbacteria, some of which are pathogens, while others are weak pathogensor saprophytes. Many biotypes of P. syringae produce one or morebioactive substances that may allow the bacteriumto survive in itsniche; for instance, on a leaf surface where it must compete with fungiand other bacteria.

A number of novel strategies for the biological control of fungal plantdiseases have been developed based on this observation. For example, atransposon-generated regulatory mutant of the wild-type strain of P.syringae 174 (MSU 16H) has been isolated that in culture producesincreased zones of fungal inhibition when compared to the wild-typebacterium.

Pseudomonas syringae MSU 16H is a transposon-generated mutant of awild-type strain that has attracted interest for its ability to make inculture elevated amounts of antifungal metabolites and to confer agreater protection than the wild-type strain in elms infected withCeratocystic ulmi, the causal agent of Dutch elm disease (Lam et al.,(1987) Proc. Natl. Sci. U.S.A. 84, 6447-6451). It was tested in elms andshown to confer a greater protective effect than the wild-type strain.More extensive tests on field-grown elms have confirmed the phenomenonof biocontrol at the prophylactic level.

Recently those metabolites, called pseudomycins, have been isolated andtheir structures have been partially characterized (Harrison et al.,(1991), J. Gen. Microbiol. 137, 2857-2865).

Identification of the chemical nature of such toxins, however, has beenvery limited. Prior to 1970 no correct toxin structure had beenpublished. More recently, attempts have been made to isolate andcharacterize the molecules responsible for the antimycotic activity ofP. syringae MSU 16 H. However, none have been successful because thebioactivity has not been preserved.

The present inventors isolate the complete structure of pseudomycins A,B, C and C'. They are new lipodepsinonapedtides related to syringomycins(Segre et al. (1989) FEBS Lett. 255, 27-31; and Fukuchi et al. (1990)Tetrahedron Lett. 31:1589-1592), syringotoxin (Ballio et al. (1990) FEBSLett. 269; 377-380; and Fukuchi et al. (1990) Agric. Biol. Chem. 54;3377-3379) and syringostatins (Isogai et al. (1990) Tetrahedron Lett.31, 695-698), a group of antimicrobial compounds produced by differentisolates of P. syringae pv. syringae.

The inventors show that pseudomycins D and D' are identical tosyringopeptin SP₂₅ -A and SP₂₅ -D respectively, lipodepeipeptides with25 amino acid residues produced by some isolates of P. syringae pv.syringae (Ballio et al. (1991) FEBS Lett. 291: 109-112).

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been discovered thatthe peptide antimycotics (pseudomycins) prepared according to thepresent invention possess broad spectrum antibiotic activity and havenumerous applications. In particular, it has been unexpectedly foundthat the peptide antimycotics of the present invention have significantantimycotic activity especially useful for the treatment of human oranimal fungal pathogens.

Furthermore, it has been discovered that the pseudomycins aresurprisingly non-toxic and may be used in therapeutic doses effectivenot only to achieve stasis but to exterminate the fungal organism inhumans or animals. In particular, the peptidyl phytotoxin exhibitsinhibitory activity against fungal pathogens such as Candida albicans,Candida tropicalis, Cryptococcus neoformans, Bipolaris spicifera andAspergillus fumigatus. The peptidyl phytotoxin is especially effectiveagainst Candida albicans. These human fungal pathogens causeopportunistic infections in immunologically compromised patients, suchas those suffering from AIDS. Very low doses are effective against thefungal pathogens, and the pseudomycin is essentially non-toxic.

Generally, for surface infections, pseudomycin may be applied in a creamor lotion and applied to the affected area. For deep systemicinfections, it is preferred that the pseudomycins be injectedintravenously.

Further in accordance with the present invention, it has beenunexpectedly discovered that successful purification of pseudomycins maybe accomplished by the elution of pseudomycins from a C-8 HPLC columnwith t-propanol in trifluoro acetic acid (TFA) as the eluant.Surprisingly, this did not appreciably decrease the activity of thecompound. TFA is used to preclude degradation of the pseudomycins. TFAalso functions as an ion pairing agent, and the use of a gradient ratherthan an isocratic system substantially reduces retention times.

The purification of pseudomycins allowed the acquisition of accuratemass spectrometry data and amino acid analysis. The result is theidentification of a novel peptidyl toxin produced by Pseudomonassyringae MSU 16H. Pseudomycins are three different peptides, pseudomycinA, pseudomycin B and pseudomycin C, each having a molecular weight of1224, 1208 and 1252 Da, respectively. Pseudomycins A-C comprise Asx,Ser, Arg, Dab and Lys in integer molar ratios of 1:1:1:3:1. (See Table20.) To obtain the ratios, the data are reduced to integral values basedon what could possibly be allowed by the molecular weights as determinedby two independent mass spectral methods--FAB and time of flight massspectroscopy. Syringomycin differs substantially from pseudomycin inamino acid composition. If one allows for the value of 11-15 as theintegral value for 1 residue per mole of pseudomycin or syringomycin,then 36-40 would represent 3 residues (as per diaminobutyric acid).Thus, only nine residues are allowed per total molecule of pseudomycinsA, B, or C and syringomycin. This is based on the fact that themolecular weights of these substances is

Ca 1200. Nine residues would bring the total contribution of the aminoacid component to 1000. This difference is contributed by a fatty acidresidue. The data of Table 20 show the fact that pseudomycins differfrom syringomycin by the lysine and aspartic acid contents. The integralratios assigned allow for only the residue number placed in parenthesisand no other value.

Thus, it is an object of the present invention to prepare treatmentmethods and formulations comprising peptide antimycotics, termedpseudomycins, for the treatment of human and animal pathogens.

It is a further object of the present invention to develop a method ofpurification for the peptide antimycotics or pseudomycins.

In particular, it is an object of the present invention to develop apurification scheme which is simple and efficient and results in highyields of active pseudomycin.

It is yet another object of the present invention to develop a method ofenhancing the growth characteristics of P. syringae and its toxinproduction.

Further, it is an object of the present invention to chemicallycharacterize and identify pseudomycins.

It is yet another object of the present invention to prepare compoundseffective in the treatment of plant disease or effective as use asherbicides.

BRIEF DESCRIPTION OF DRAWINGS

Reference is now made to the drawings accompanying the applicationwherein:

FIG. 1 is a RP-HPLC elution profile of partially purified pseudomycinfrom the Amberlite XAD-2 column.

FIG. 2 is a RP-HPLC elution profile of pseudomycin run on a lineargradient of 0-80% acetonitrile over 20 minutes.

FIG. 3 shows thin layer chromatography of pseudomycin.

FIG. 4 shows FAB mass spectra of purified pseudomycin B (M+H=1208).

FIG. 5 shows ¹ H-NMR spectrum of pseudomycin.

FIG. 6 is a spectrophotometric scan of pseudomycin dissolved in 0.1%TFA.

FIG. 7 is a growth curve for P. syringae 16H grown in still culture inPDB and shake culture in mDyes medium.

FIG. 8 shows HPLC purification of pseudomycins A-D including a compositechromatograph of the four single peaks in the second run at inset.

FIG. 9 shows CE of syringomycin and pseudomycins.

FIG. 10 is an analysis of Dab and Phe by CE.

FIG. 11 shows Reverse phase HPLC of a P. syringae MSU 16H extractpartially purified produced an elution pattern of the same type observedwith syringomycin and syringotoxin-producing strains.

FIG. 12 shows the complete structure of pseudomycin A.

FIGS. 13 and 14 show the complete chemical formula and molecularstructure of pseudomycin A.

FIG. 15 shows a capillary zonal electrophoresis (CZE) of pseudomycins A,B, C and syringomycin.

STATEMENT OF DEPOSIT

P. syringae MSU 16H is publically available from the American TypeCulture Collection, Parklawn Drive, Rockville, Md. as Accession No. ATCC67028. During the pendency of this application, access to the depositwill be forwarded to one determined by the Commissioner to be entitledthereto. All restrictions imposed by the depositor on the availabilityto the public of the deposited material will be irrevocably removed uponthe granting of the patent. The deposit will be maintained for a periodof at least thirty years or at least five years after the most recentrequest for the furnishings of a sample of the deposited material. Thedeposit will be replaced should it become necessary due to inviability,contamination or loss of capability to function in the manner describedin the specification.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, Pseudomonas syringae MSU 174was isolated from a Montana barley field. In an attempt to increaseantimycotic production, MSU 174 was mutagenized by transposonmutagenesis using TN 905. A "super producer" was isolated and calledstrain 206. Strain 206 was labeled P. syringae MSU 16H and used in thepurification of pseudomycin. A culture of P. syringae is on deposit atMontana State University and available from the American Type CultureCollection. Other sources of pseudomonas syringae known in the art mayalso be used. P. syringae MSU 16H was stored in 15% glycerol at -70° C.

The growth characteristics of P. syringae and its antimycotic (toxin)production in the absence of its host are determined. This is preferablyassayed with a growth curve, the time of maximum antimycotic toxinproduction, and possible induction of antimycotic production bycompounds from plants or fungi affected by the antimycotic.

Some antimycotic producing plant pathogenic fungi lose their ability toproduce their antimycotic in the absence of the host plant orconstituents of the host plant. In other cases there may be someconstitutive production of the antimycotic, but the amount produced isincreased in the presence of plant material. This phenomena has alsobeen observed with some, but not all, strains of P. syringae.

In order to determine the most advantageous method for culturing P.syringae, the following was performed: Data for a growth curve wasgathered for modified Dyes media. Two tubes, each with 3 ml of mDyes wasinoculated with a single colony of P. syringae MSU 16H then grown inshake culture (180 rpm) at room temperature, overnight. After 24 hours,these cultures contained ≈6×10⁷ org./ml and were used to inoculate twoside arm flasks containing 250 ml of mDyes. The bacteria were grown inshake culture, at room temperature, for a total of six days. At timedintervals (0.5, 1, 2, 4, 8, 24, 48, 72, and 96 hours) Klett readingswere taken on a Klett-Summerson model 800-3, photoelectric colorimeter.Klett readings were averaged then recorded as Klett units versus time(hours). The instrument was zeroed with uninoculated media.

The same procedure was repeated to obtain a growth curve with potatodextrose in broth (PDB) in still culture.

Growth of P. syringae MSU 16H followed the "normal" sigmoidal growthpattern when grown in mDyes medium. With reference to FIG. 7, thebacteria remained in lag phase for up to 24 hours at which time theyentered logarithmic growth. Growth tapered off and entered stationaryphase at about 96 hours. A similar growth pattern resulted with PDB,however, the bacteria reached log phase and stationary phase later, 36hours and 144 hours respectively (FIG. 7). The PDB culture also reacheda higher density than the mDyes culture.

As one of ordinary skill in the art will appreciate, it is sometimesadvantageous to use a minimal media when growing cultures for naturalproduct isolation, to minimize extraneous compounds requiring separationfrom the natural product. A preferred media comprises potato dextrose inbroth (PDB). The dyes media may be supplemented with FeCl₃, ornithine,and histidine. In a most preferred embodiment, cells are grown in potatodextrose in broth (PDB) as a still culture at 23° C. for 6 days.

P. syringae may also be cultured in other media such as Dyes salts witheither 1.5% glucose, 0.5% yeast extract (YE), or 2% proteose peptone #3(PP) wt/v), and/or with the addition of arbutin or geotrichum.

To examine antimycotic production over time, fifteen 100 ml aliquots ofPDB, were inoculated with a 3 ml overnight culture of P. syringae. Thecultures were grown in triplicate as still cultures at room temperaturefor 0, 3, 6, 9, and 12 days before fractionation by acetoneprecipitation. Each fraction was dried under nitrogen and weighed. Thepreparation was resuspended in 50% 1-propanol with 0.1% TFA atconcentrations of 500, 50, 5, 0.5 and 0.05 μg/μl. Each of these (10 μl)was spotted onto PDA and bioassayed (Table 1).

The presence of antimycotic was detected as early as 6 days when a totalof 500 μg was spotted (10 μl of 50 μl/μl). The activity seemed to remainthe same until the termination of the experiment at 12 days. Withreference to FIG. 7, the bacterial culture entered the stationary phaseof growth at 6 days.

                  TABLE 1                                                         ______________________________________                                        Production of pseudomycin over time. (+) = zone                               of inhibition, (-) = no zone of inhibition.                                                 Concentration (μg/μl)                                     Day   Dry Wt. (g/100 ml)                                                                          500     50   5    0.5   0.05                              ______________________________________                                        0     2.32 +/- .01  -       -    -    -     -                                 3     1.67 +/- .09  -       -    -    -     -                                 6     1.36 +/- .47  +       +    -    -     -                                 9     1.10 +/- .18  +       +    -    -     -                                 12    1.01 +/- .30  +       +    -    -     -                                 ______________________________________                                    

Purification of pseudomycin is challenging due to the potential loss ofbiological activity at various stages of purification. Conditions suchas pH, heat stability, and solvent solubilities must be considered. Inaddition, there are problems such as ionic exchange or nonspecificirreversible binding to glass, silica, or even column packing material.

Prior art problems with instability were corrected by using compatiblesolvents and maintaining a low pH by adding TFA to all the solvents.

Acetone precipitation, followed by column chromatography with thenonionic adsorbent, Amberlite XAD-2 column, proved to be a satisfactorymethod of purification. A typical problem with the separation ofpeptides by RP-HPLC is exceedingly long retention times. However,according to the present invention, this problem is solved by theaddition of the ion pairing agent TFA, and the use of a gradient ratherthan an isocratic system.

In accordance with the present invention, pseudomycin may be initiallypurified by either butanol extraction or acetone precipitation. Acetoneprecipitation is the preferred embodiment.

Butanol Extraction

Cultures are centrifuged, preferably at 5000×g for 10 minutes to pelletthe cells. The supernatant liquid is flash evaporated, preferably to 200ml at 38° C., then extracted about three times with an equal volume of1-butanol. The butanol fraction may be extracted with water, then thebutanol phase flash evaporated to dryness at 38° C. The driedconcentrate may be resuspended in methanol for transfer, then driedunder nitrogen for storage at -20° C.

Acetone Precipitation

Cultures are preferably mixed 1:1 (v/v) with acetone then centrifuged at5000×g for 10 minutes. The liquid supernatant is flash evaporated to 200ml, then brought to a final concentration of 60% acetone. This may beallowed to precipitate overnight at 4° C. with gentle stirring. Theprecipitate is removed by centrifugation at 5000×g for 10 minutes. Theliquid supernatant is flash evaporated to dryness.

The preferred method of further pseudomycin purification is by elutingthe sample through a column. A typical problem with the separation ofpeptides by RP-HPLC is exceedingly long retention times. However,according to the present invention, this problem is solved by theaddition of the ion pairing agent TFA, and the use of a gradient ratherthan an isocratic system.

Generally, Amberlite XAD-2 (mesh size 20-60) is washed with 0.1% TFA,then packed in a 1.6×40 cm column. The column is equilibrated with 0.1%TFA. The sample prepared by either method above (butanol or acetoneprocedure) is preferably suspended in one liter 0.1% TFA and loaded ontothe column at 1 ml/min with a Waters M25 solvent delivery system.Pseudomycin is preferably eluted with a nonlinear gradient of 1 to 100%1-propanol with 0.1% TFA and the column eluate monitored at 254 nm. Thegradient former is preferably a Kratos Spectroflow 430. Fractions may becollected with a Gilson microfractionator and tested for bioactivity,preferably antifungal activity. Active fractions are pooled, dried witha rotary evaporator, and resuspended in 50% 1-propanol containing 0.1%TFA, and filtered through a 0.2 micrometer Millipore filter.

Final drying of small quantities for analysis before storage at -20° C.may be accomplished by placing the sample under a flow of nitrogen gas.The sample (10 microliters) was applied to a PDA plate and allowed todry. The plate was oversprayed with a sterile water suspension ofGeotrichum candidum, sealed with parafilm, and incubated at roomtemperature. Zones of complete inhibition were noted.

Active fractions from the Amberlite column, after filtering through a0.2 micron filter (Gelman Sciences) are subjected to reverse phase highperformance liquid chromatography (RP-HPLC) on a 4.6×100 mm C8 column(Amicon MC-250) with a 1-propanol, nonlinear gradient (0 to 30%1-propanol, o.1% TFA, Waters #2 gradient over 35 minutes). The flow rateof the mobile phase is preferably 1 ml/min. Each peak is collected,evaporated, and resuspended in 50% 1-propanol with 0.1% TFA before beingbioassayed. Single active peaks are combined then reinjected for furtherpurification.

Collections from a single peak are preferably again subjected to RP-HPLCutilizing a second solvent system. The samples are eluted with a linear,acetonitrile, 0.1% TFA gradient (Waters #6, 0 to 80% acetonitrile, 0.1%TFA) over a 20 minute period.

Purification of larger quantities of pseudomycin may be performed on a10×250 mm Amicon MC-250 C8 preparative column with a flow rate of 2ml/min.

With both columns, a Waters system should be used including a model 440absorbance detector monitoring at a wavelength of 254, and a model 660solvent programmer. Two solvent delivery systems are highly preferred, amodel M45 and a model A6000.

Collections from each peak to be used for biological testing may beflash evaporated and resuspended in 50% 1-propanol containing 0.1% TFA.Samples for mass spectrometry may be flash evaporated then concentrated,but not dried, under nitrogen. Samples for amino acid analysis may becollected from the HPLC directly into polypropylene cryovials, andassayed directly without further concentration.

FAB Mass Spectrometry

Fast atom bombardment (FAB) mass spectrometry may be performed in orderto determine the molecule weight of the pseudomycin. Mass spectrometryutilizing electron bombardment for peptides requires chemicaldegradation and derivatization, while FAB mass spectrometry makes itpossible to analyze underivatized large polar biomolecules.

Analysis is preferably carried out with a VG MassLab Trio2 automatedmass spectrometer connected to a Hewlett Packard model 5890 gaschromatograph. The column is preferably a 30 meter P-5 microcolumn. Thesample may be run in both a glycerol and thioglycerol matrix.

Nuclear Magnetic Resonance

The proton nuclear magnetic resonance (NMR) spectrum may be recorded ona 500 MHz Bruker spectrometer. Chemical shifts are preferably recordedin ppm units relative to trimethylsilane (0 ppm) with D₂ 0.

Absorbance Characteristics

Pseudomycin, dissolved in 0.1% TFA, may be scanned on a Beckman DU-50Spectrophotometer. The solution (100 μl) is weighed on a Cahn model Gelectrobalance to obtain a value for the calculation of the molarconcentration. The molar extinction coefficient can be calculated usingthe maximum absorbance and the molar concentration. The scan andweighing are preferably repeated three times and the average resultrecorded.

Thin Layer Chromatography

The RP-HPLC sample of pseudomycin (3 μg) may be spotted onto a silicagel 60 F254 thin layer plate (E. Merck Science). The same amount ofsyringomycin may be spotted adjacent to the pseudomycin for acomparative standard. TLC plates are preferably run in three separatesolvent systems; 1) 1-butanol:pyridine:acetic acid:water, 2)1-butanol:2-picoline:acetic acid:water, and 3)1-butanol:2,6-1utidine:acetic acid:water. The ratio should be the samefor each system, 15:10:3:12, (v/v). TLC plates are then sprayed withninhydrin reagent; 0.5% ninhydrin in 95% ethanol (v/v). The compoundswill appear as light purple spots.

Purity is mandatory for the proper identification of an unknowncompound. A single peak upon elution from the HPLC column is suggestiveof purity but in itself is not enough. Thin layer chromatography (TLC)is one method for examining purity. Peptides are markedly hydrophiliccompounds and are only slightly soluble in nonaqueous solvents. Solventsystems used in their separation must generally contain water.

Capillary Electrophoresis (CE)

This is preferably performed on an Applied Biosystems model 270ainstrument. Samples of the antimycotics may be loaded into a 50 μm×72 cmfused silica capillary by vacuum aspiration, then electrophoresed at 25kV, 24 μA, 38° C., in 50 mM-acid. Peaks are detected at 200 nm. Theabsorbance of the effluent should be monitored at 254 nm.

Amino Acid Analysis

For the amino acid analysis, a model 420 derivatizer coupled to a model130 HPLC system is preferably utilized. Data may be acquired andanalyzed by a model 920 software system. Amino acid sequencing may beperformed with a model 477 sequencer with one line PTH aminophenylthiohydantoin (PTH) amino acid analysis. Peak quantitation may beperformed by software supplied with the model 477 sequencer. Further,peak identification may be performed manually by comparison to standardchromatograms.

Heat Stability

The heat stability of the pseudomycin may be analyzed according to thefollowing method: A 1-butanol crude extract (75 μl) of pseudomycin, at aconcentration of 33 μg/μl, are preferably placed into each of eight 1.5ml Eppendorf tubes (or reactivials for the 100° C. test). Tubes are thenincubated preferably at each of four separate temperatures (e.g. 15°,30°, 60°, and 100° C.) for a period of time, preferably six days.Samples (5 μl) may be removed from each tube at 0, 0.5, 1, 2, 4, 8, 24,48, 72, and 96 hours and spotted on a PDA plate. After drying, theplates are oversprayed with G. candidum, then incubated at roomtemperature overnight. Inhibition was recorded as plus or minus at eachtime and temperature.

pH Sensitivity

The pH sensitivity of the pseudomycin may be analyzed according to thefollowing method: a 1-butanol crude extract (5 μl), at a concentrationof 100 mg/ml, is dissolved in 200 μl of the treatment solution (Table2). The concentration of each buffer may be 0.01M. Each buffer isadjusted to a pH equal to its pK_(a) with 1M sodium hydroxide or 1Mhydrochloric acid. The samples are incubated at room temperature for aperiod of time, preferably four days. At 0, 1, 2, 3, and 4 days, eachsolution (10 μl is spotted onto PDA and bioassayed. All treatments maybe run in duplicate. The positive control is the 1-butanol extractdissolved in water and methanol. The negative controls are the bufferswithout the extract.

                  TABLE 2                                                         ______________________________________                                        Buffers for testing pH sensitivity of the                                     antimycotic.                                                                          Buffers     pH                                                        ______________________________________                                        1.        Citric Acid   3.06                                                  2.        Citric Acid   4.75                                                  3.        Ammonium Acetate                                                                            4.75                                                  4.        Citric Acid   5.40                                                  5.        Pipes         6.80                                                  6.        Tris          8.00                                                  7.        Tris          9.00                                                  8.        Boric Acid    10.00                                                 ______________________________________                                    

The stability of the pseudomycin in various solvents may be analyzedaccording to the following method: A 1-butanol crude extract (5 μl at 33μg/μl) is added to 100 μl of solvent. Each solvent (100 μl) alone isused for the negative control. The solutions are incubated at roomtemperature for 24 hours, at which time they are dried under nitrogenthen resuspended in 15 μl of methanol and 10 μl are removed forbioassay.

The solvents tested may be: 1-propanol, (50% and 100%); TFA, (1, 2, and3M); HCL, (0.5, 1, 2, and 4M); 0.1% TFA, 10% acetonitrile; 0.1% TFA, 90%acetonitrile; 100% 1- butanol; 100% methanol; 100% water; and 0.1% TFA.

Before a protocol for antimycotic purification can be adopted, a goodbioassay must be developed which is preferably both sensitive andquantitative. The overspray method utilizing G. candidum is the methodof choice because of the availability of the organism. Fractionsresulting from the various purification steps are spotted onto PDA andallowed to dry. The plate is oversprayed with a sterile water suspensionof the fungus then incubated overnight at room temperature. Presence ofantimycotic is indicated by a clear zone of inhibition. Weak zones ofinhibition, that eventually overgrew with mycelia, are considerednegative.

Known amounts of crude extract are spotted for assay on separate PDAplates. Great variation of zone size is noted depending on the age ofthe plate and the amount the solution spread on the plate. To reducevariation, the samples are preferably applied to antibiotic filter discswhich are then laid on the agar plate before overspraying. Theantimycotic adsorbed to the disc, preventing diffusion onto the agarplate. Results in the bioassays are recorded as plus or minus.

With regard to the purification of pseudomycin, solvents used during thepurification procedures could affect the activity of the antimycotic. Inaccordance with the present invention, 165 μg of 1-butanol crude extractwas added to 10 μl of the solvent to be tested. Each vial was incubatedfor 24 hours at room temperature then dried under nitrogen andresuspended in 15 μl of methanol. An aliquot (10 μl) was removed fromeach vial and bioassayed. Each solvent was tested without the crudeextract as a negative control. Pseudomycin remained active in allsolvents tested except the 4M HCl (Table 3). TFA at 3M, and theremaining concentrations of HCl, also decreased the zones of inhibition.

                  TABLE 3                                                         ______________________________________                                        Sensitivity of toxin to different solvents. (-) =                             no zone of inhibition (+) = a zone of inhibition <1 cm.                       dia. ++ = a zone of inhibition > or = 1 cm. dia.                              Solvent            Treatment Control                                          ______________________________________                                        100% 1-propanol    ++        -                                                50% 1-propanol     ++        -                                                3M TFA             +         -                                                2M TFA             ++        -                                                1M TFA             ++        -                                                0.5M TFA           ++        -                                                0.1% TFA, 10% acetonitrile                                                                       ++        -                                                0.1% TFA, 90% acetonitrile                                                                       ++        -                                                0.5M HCl           +         -                                                1M HCl             +         -                                                2M HCl             +         -                                                4M HCl             -         -                                                100% 1-butanol     ++        -                                                100% methanol      ++        -                                                100% water         ++        -                                                100% water, 0.1% TFA                                                                             ++        -                                                ______________________________________                                    

Other solvents may be tested in a similar manner.

Cells were grown for six days in still culture in PDB at roomtemperature. The culture was mixed 1:1 (v/v) with acetone thencentrifuged to remove cellular debris. The supernatant was first flashevaporated to 200 ml, then acetone was added to 60% (v/v). The mixturewas allowed to precipitate overnight. Precipitate was removed bycentrifugation. Antimycotic activity remained in the supernatant liquid.

The supernatant liquid of the PDB media was concentrated, thenresuspended in one liter of 0.1% trifluoroacetic acid (TFA), and loadedonto an Amberlite XAD-2 column. After elution with a propanol gradient,the fractions were bioassayed. With reference to FIG. 8, the pseudomycinfraction from Amberlite XAD-2 chromatography was resolved into its fourcomponents by reversed-phase HPLC using a C-8 column and a hyperbolicgradient of 10 propanol in TFA. Letters refer to the respectivepseudomycins, A-D. Each pseudomycin fraction was then rechromatographedon the same column, but was eluted with a linear gradient ofacetonitrile in TFA. A composite chromatograph of the four single peaks,and the retention times (min), observed in these second runs ispresented in the inset.

Positive fractions were combined, filtered, and subjected to reversephase high performance liquid chromatography (RP-HPLC). Two differentsolvent systems were utilized; 1) a 1-propanol nonlinear gradient, and2) an acetonitrile linear gradient. Propanol was the solvent of choicefor the separation of peptides and proteins because the concentrationsneeded for elution are lower than with other organic modifiers, reducingthe chance of activity loss. Acetonitrile is a good choice for the samereasons. TFA (0.5%) is necessary for adjustment of the pH and acts as anion pairing agent. TFA modifies the polarity of the peptide throughion-pair formation which leads to an increase in retention time andtherefore, better separation. Volatility of TFA is also a positiveaspect when considering amino acid analysis and sequencing.

The propanol gradient was a nonlinear (Waters #2) gradient of 0 to 30%1-propanol, 0.1% TFA over 35 minutes. Each peak from the propanolgradient was collected, concentrated by flash evaporation, then testedfor bioactivity. With reference to FIG. 1, peaks 3, 4, and 6 were foundto be active. The eluate collected from 16 to 20 minutes (labeled #5 inFIG. 1) was also active but no definite peak was seen on the elutionprofile. The retention times are listed in Table 4. These peaks werereinjected for further purification.

                  TABLE 4                                                         ______________________________________                                        Retention times of peaks from the HPLC after                                  elution with a nonlinear propanol gradient.                                   Peak number    retention time                                                 ______________________________________                                        3              13 min. 30 sec.                                                4              14 min. 45 sec.                                                5              --                                                             6              22 min.  0 sec.                                                ______________________________________                                    

The final step was to subject the eluate from peak #4 to the RP-HPLCwith the linear acetonitrile gradient, 0 to 80% acetonitrile over 20minutes. The retention time was 14 minutes and 45 seconds (FIG. 2). Thisprocedure yielded an average of 0.0015 grams of purified toxin ascompared to 0.0002 grams with the butanol procedures. Both the 1-butanolextraction method and the acetone precipitation method resulted in theisolation of the same compound as confirmed by thin layer chromatographyand mass spectrometry data.

                  TABLE 5                                                         ______________________________________                                        Purification table for the acetone                                            precipitation procedure                                                       Stage of      Grams of Dry Weight                                                                          Purification                                     Purification  per liter      Fold                                             ______________________________________                                        PDA culture   27.6           1                                                1.sup.st acetone ppt.                                                                       15.0           1.8                                              2.sup.nd acetone ppt.                                                                       12.8           2.2                                              Amberlite XAD-2                                                                             0.016          1,725                                            RP-HPLC       0.0015         18,400                                           ______________________________________                                    

As a preliminary check, the eluate collected at the time of each peakfrom the propanol gradient, was subjected to FAB mass spectrometry. HPLCpeak number 4 was the only peak that appeared to be pure, having asingle mass ion peak. Mass spectra data from 3 and 5 had more than onepeak, one of which had the same mass ion assignment as the peak fromHPLC peak number 45. Spectral data for HPLC peak number 6 also showedseveral peaks but none at the same mass as HPLC peak number 4. It isbelieved that this may suggest a second antimycotic compound in thesepreparations.

PDB alone was carried through the same purification steps as the P.syringae culture. There were no detectable antimycotics present in PDBalone.

Confirmation of Purity--Thin Layer Chromatography

Butanol:pyridine:acetic acid:water (15:10:3:12) was the solvent ofchoice. The RP-HPLC sample (3 μg) was spotted on to a silica gel TLCplate. Syringomycin was also spotted on the plate for a comparativestandard. Both compounds migrated as single spots with R_(f) 's of 0.59and 0.37, respectively (FIG. 3). Not only did pseudomycin appear pure,but it migrated differently than syringomycin. To further establishpurity, pseudomycin was run in two additional solvent systems; picolineor lutidine was substituted for pyridine. These solvents are methylatedanalogs of pyridine. Again single spots were apparent with R_(f) valuesas indicated in FIG. 3.

Chemical Characterization

The purified sample was subjected to Fast Atom Bombardment (FAB) massspectrometry. The sample was run in two different matrices, glycerol andthioglycerol. The mass H+ was found to be 1208 (FIG. 4). The same samplewas used for amino acid analysis and sequencing. Preliminary results forpseudomycin indicate the presence of seven amino acids; aspartate,serine, lysine, phenylalanine, and arginine (1:1:1:3:1) and one unknownamino acid. The amino acid analysis of syringomycin is arginine,phenylalanine, serine, and 2,4-diaminobutyric acid (1:1:2:2).

Using post column derivatization, with the Applied Biosystemsinstrumentation, the fourth amino acid in syringomycin,2,2-diaminobutyric acid, is undistinguishable from phenylalanine. The ¹H-NMR spectrum (FIG. 5) indicates the presence of protons on an aromaticring in the range of 7.1-7.4 ppm which may be from phenylalanine. Thissuggests that phenylalanine is one of the amino acids but does not ruleout the presence of 2,3-diaminobutyric acid. The sum of the molecularweights of the suspected amino acids is considerably lower than the masscontained from the FAB mass spectrometry data. This may be due to thepresence of a long chain fatty acid attached to the peptide, similar tothe one found in syringomycin.

Pseudomycin, dissolved in 0.1% TFA, was scanned using aspectrophotometer. There were two peaks of absorbance (FIG. 6); one at254 nm and one at 280 nm. The solution (100 μl) was weighed on anelectrobalance. The molar extinction coefficient was calculated for eachwavelength; Am₂₅₄ =167, Am₂₈₀ =146.

Heat stability was tested at four different temperatures; 15°, 30°, 60°,and 100° C. (Table 6). Butanol crude extract (75 μl) of pseudomycins, ata concentration of 33 μg/μl placed into each of eight 1.5 ml Eppendorftubes. Tubes were then incubated at each of four temperatures (15°, 30°,60° and 100° C.) for six days. Throughout the incubation period, samples(5 μl) were removed from each tube at 0, 0.5, 1, 2, 4, 8, 24, 48, 72 and96 h and spotted on a PDA plate. After drying, the plates wereoversprayed with G. candidium (10⁶ cells ml⁻¹), then incubated at roomtemperature overnight. Zones of growth inhibition were recorded as ++(>2cmJ), +(<2 cm), or--(no inhibition). The experiment was repeated twicewith identical results. There was total loss of activity by the fourthday. When the extract was incubated at 100° C. there was a largedecrease in the zone of inhibition for antimycotic heated 30 minutes anda complete loss of activity for antimycotic heated four hours. Thisantimycotic demonstrates considerable heat stability.

                  TABLE 6                                                         ______________________________________                                        Heat stability of the toxin. (+) = zones of                                   inhibition <2 cm. (++) = zones of inhibition > or = 2 cm.                     (-) = no apparent zone of inhibition.                                         Temp.  Time (hours)                                                           °C.                                                                           0      0.5    1    2    4    8   24   48  72  96                       ______________________________________                                        15     ++     +      ++   ++   +    +   ++   +   +   +                        30     ++     ++     ++   ++   ++   +   ++   +   +   +                        60     +      +      +    +    +    +   +    +   +   +                        100    ++     +      +    +    -    -   -    -   -   -                        ______________________________________                                    

Partially purified pseudomycin (5 μl), at a concentration of 100 mg/ml,was dissolved in 200 μl of water, methanol (positive control), and eachof eight different buffers (0.01M). In addition, one tube of eachtreatment without the extract was prepared as a negative control. Eachbuffer was adjusted to a pH equal to its pK_(a). Each solution wasincubated at room temperature. After 0, 1, and 2 days, 10 μl of eachsolution was spotted onto PDA and bioassayed. Pseudomycin remainedactive in the water, methanol, and the buffers at or below a pH of 4.75(Table 7). Activity in buffers at or above pH 5.4 was apparent initiallybut the zones of inhibition were overgrown in two days. The staticinhibition was also noted in the same control treatments and may be dueto high pH values. These were recorded as negative results.

                  TABLE 7                                                         ______________________________________                                        Sensitivity of activity to pH changes.                                        ______________________________________                                                  Treatments    pH                                                    ______________________________________                                        1.        Citric Acid   3.06                                                  2.        Citric Acid   4.75                                                  3.        Ammonium Acetate                                                                            4.75                                                  4.        Citric Acid   5.40                                                  5.        Pipes         6.80                                                  6.        Tris          8.00                                                  7.        Tris          9.00                                                  8.        Boric Acid    10.00                                                 ______________________________________                                        Results                                                                       Day     1     2       3   4     5   6     7   8                               ______________________________________                                        0       +     +       +   +     +   +     +   +                               1       +     +       +   +     +   -     -   -                               2       +     +       +   +     +   -     -   -                               3       +     +       +   +     +   -     -   -                               4       +     +       +   +     +   -     -   -                               ______________________________________                                    

Table 8 below summarizes the effects of solvent and pH on theantimycotic activity of pseudomycins.

                  TABLE 8                                                         ______________________________________                                        Effects of solvent and pH on the antimycotic                                  activity of the pseudomycins                                                              Antimycotic                                                       Solvent     activity*  Buffer      Inhibition†                         ______________________________________                                        100% 1-butanol                                                                            ++         Citrate, pH 3-1                                                                           +                                          50% 1-propanol                                                                            ++         Citrate, pH 4-8                                                                           +                                          100% 1-propanol                                                                           ++         Acetate, pH 5-4                                                                           -                                          100% methanol                                                                             ++         PIPES, pH 6-8                                                                             -                                          100% water  ++         Tris, pH 8-0                                                                              -                                          0-1% TFA    ++         Borate, pH 9-0                                                                            -                                          10% CH.sub.3 CN in 0-                                                                     ++         Borate, pH 10-0                                                                           -                                          1% TFA                                                                        90% CH.sub.3 CN in 0-                                                                     ++                                                                1% TFA                                                                        0-5M-TFA    ++                                                                1M-TFA      ++                                                                2M-TFA      ++                                                                3M-TFA      +                                                                 0-5M-HCl    +                                                                 1M-HCl      +                                                                 2M-HCl      +                                                                 4M-HCl      -                                                                 ______________________________________                                         *A 1butanol extract of pseudomycins (5 μl at 33 μg μl.sup.-1) wa     added to 100 μl of each solvent. Each solvent (100 μl) was used as      negative control. The solutions were incubated at 23° C. for 24 h,     then dried under N.sub.2 and resuspended in 15 μl methanol. Each           methanol extract (10 μl) was then spotted on a PDA plate, after which      the plate was dried, oversprayed with G. candidum (10.sup.6 cells             ml.sup.-1), and incubated at room temperature overnight. Zones of growth      inhibition were recorded as ++ (>2 cm), + (≦2 cm), or - (no            inhibition). No inhibition of fungal growth was observed with any of the      controls (data not shown).                                                    †Duplicate 5 μl aliquots of a 1butanol extract of pseudomycin       (100 μg μl.sup.-1) were dissolved in 200 μl of 10 mMbuffer. Afte     incubation for 48 h at room temperature, a 10 μl aliquot was bioassaye     as described above. Zones of growth inhibition were recorded as +             (≦2 cm) or - (no inhibition). Positive controls, using methanol        instead of buffers, showed clear zones of inhibition. Negative controls,      using buffers alone, showed no inhibition. The experiment was repeated        twice with essentially the same results.                                 

Purity of Pseudomycins

Capillary electrophoresis (CE) and TLC were used in addition to HPLC toassess the purity of the pseudomycin preparations. In addition, sinceother strains of P. syringae produce syringomycin, an antimycotic withchromatographic properties similar to those of the pseudomycins, CE andTLC was used to determine whether syringomycin was identical to one ofthe pseudomycins.

Pseudomycins A, B, and C each produced a single, sharp, major peak inCE; these peaks represented 95% 94%, and 95% respectively, of the totalUV-absorbing material present in each fraction. Syringomycin alsodisplayed a single, predominant peak, containing 83% of the totalUV-absorbing material in the preparation. All five samples were thenelectrophoresed together to determine if any co-migrated. All had uniqueelectrophoretic mobilities (FIG. 9). Thus each of the pseudomycins ofthe invention has a purity of about 95%.

With regard to FIG. 9, approximately 3 ml each of syringomycin andpseudomycins A-C were loaded into the capillary. Electrophoresis wasperformed as specified previously. The abscissa representselectrophoretic migration time, measured from the anodic end of thecapillary to the detector. Peaks are denoted as follows: S.syringomycin; A, B and C, pseudomycins A, B, and C and a minor componentfound with D, respectively.

To ensure that loading order did not affect migration times, the pairsof pseudomycins A plus B and A plus C were re-electrophoresed afterloading in both possible orders. The relative electrophoretic mobilitiesfor the three samples remained constant. The mobility differences ofsyringomycin and D relative to the triplet of A, B and C are so largethat loading order would not affect their relative positions.

TLC experiments yielded analogous results. Each of the pseudomycins andsyringomycin migrated as a single spot with a unique R_(f), value ineach of the different TLC systems. In all cases, syringomycin migratedwith a higher R_(f) than did the pseudomycins.

Amino Acid Analysis

Amino acid compositions were determined using an Applied Biosystemsmodel 420A/130A derivatizer-analyzer. Briefly, approximately 200-900pmol of each sample mixed with Inmol L-norleucine, was dried in vacuo inseparate 6×50 mm glass tubes. These tubes were then sealed, in vacuo,inside 18×150 mm Pyrex culture tubes containing 300 μl 6M-hydrochloricacid (Pierce Chemical Co.). Gas-phase hydrolysis was performed byheating at 170° C. for 30-45 minutes. After cooling, each sample tubewas removed from the ignition tube and dried in a Speed-Vac concentrator(Savant).

Hydrolysis products were dissolved in 15 μl 0.025% (w/v) K₃H-ethylenediaminetetra-acetate (EDTA) and spotted onto the glassreaction slides of the model 420A/130A instrument.Phenylthiocarbamoylation and reverse-phase HPLC were performedautomatically by the 420A/130A instrument, essentially according to themanufacturer's instructions. "Dab" refers both to Phe and Dab(diaminobutyric acid), since these molecules co-elute from the HPLC."Dab" peaks were collected and dried down prior to CE. Moles of eachamino acid were first determined using molar absorptivity values derivedfrom hydrolysed amino acid standards, then were normalized using theinternal standard L-norleucine. 3-Hydroxyaspartic acid eluted in aregion containing an injection artifact, prior to any of the other aminoacids.

Protein Sequence Analysis

Sequencing was done on polybrene-coated glass fibre supports using anApplied Biosystems model 477A/120A pulsed liquid sequenator, essentiallyas suggested by the manufacturer. Phenylthiohydantoin amino acidanalysis was performed on-line and sequence assignments were made byvisual inspection of chromatograms.

Plasma Desorption Mass Spectrometry (PDMS)

Spectra were acquired using a BioIon 20 instrument (BIO-ION Nordic; nowa division of Applied Biosystems), according to the manufacturer'sinstructions. Samples were dissolved in 0.1% TFA/50% ethanol (in water),dried on nitrocellulose-coated aluminized mylar supports, washed brieflywith water, then analyzed. Alkaline hydrolyses were done by incubating2-5 μl (about 0.1-1 nmol) of sample (from the acetonitrile/C-8 HPLCstep) with 20 μl 1% (v/v) ammonium hydroxide (in water) containingeither 50% (v/v) ethanol or 50'5 (v/v) n-propanol, for 20 minutes at 22°C. The samples were then dried in a Speed-Vac concentrator, dissolved in0.1% TFA/alcohol (the same alcohol as was used for hydrolysis), andprepared for PDMS as described above. See Table 9.

                  TABLE 9                                                         ______________________________________                                        Mass spectral analysis of pseudomycins                                        The pseudomycins (Pseudo. A-D) and syringomycin were                          analysed by PDMS, as described in Methods. Masses are those of                the most predominant (M + H).sup.+  ions. Samples were run both               without being hydrolysed and after alkaline hydrolysis in either              ethanol or 1-propanol.                                                                Pseudo.  Pseudo.  Pseudo.                                                                              Pseudo.                                                                              Syringo-                              Treatment                                                                             A        B        C      D      mycin                                 ______________________________________                                        None    1224-7   1208-6   1252-6 2401-0 1226-6                                NH.sub.3 +                                                                            1270-4   1253-3   1296-9 2384-5 1272-0                                C.sub.2 H.sub.5 OH                                                                    1243-5   1226-1   1270-1        1243-3                                                                        1208-4                                NH.sub.3 +                                                                            1284-7   1264-3   1311-7 2384-9 1284-1                                C.sub.3 H.sub.7 OH                                                                    1242-2   1226-6   1269-8        1243-0                                ______________________________________                                    

After treatment, pseudomycins A, B and C, as well as syringomycin,produced (M+H)⁺ ions shifted higher in mass by approximately 17-19 Da,equivalent to one molecule of ammonia or water. A second family of ionswas also observed, in which masses were heavier by approximately 44-46Da, equivalent to one molecule of ethanol (46 Da). These data areconsistent with a process in which the lactone is opened to an acid,amide or ester form. Additional evidence for this was obtained when1-propanol, instead of ethanol, was used in the solvent for hydrolysisand dissolution. In this case, a different high-mass family was observedapproximately 60 Da larger than that seen with the untreated molecules(Table 9). This increase is equivalent to the mass of 1-propanol.Pseudomycin D, on the other hand, lost approximately 16 Da followingboth base treatments. This difference may represent loss of oxygen (16Da), or substitution of hydroxide for chloride (17 Da) as may occur, forexample, at alkaline pH with chlorothreonine. The behavior ofpseudomycin D, which is inconsistent with its being a lactone, alonewith its molecular mass, distinguish it from other pseudomycins.

Ions with masses corresponding to those of the native molecules, ofdehydrated molecules, of dimeric molecules, and of unidentifiedmolecules were also observed but at significantly lower levels thanthose of the +18, +46 and +60 Da families.

Amino Acid Composition of Pseudomycins

Amino acid analysis were performed on the pseudomycins and syringomycinas described above. Pseudomycins A, B and C had almost identicalcompositions, containing Asx, Ser, Arg, "Dab" and Lys in the integermolar ratios 1:1:1:3:1, respectively (Tables 10 and 20). The unusualamino acid 3-hydroxyaspartic acid was also detected in each of the threemolecules. Syringomycin contained Ser, Arg and "Dab", in the integermolar ratios of 2:1:3, as well as 3(OH)Asp. Significant amounts ofaspartic acid or theonine were not detected in syringomycin. Thecomposition of pseudomycin D was more complex. Like the otherpseudomycins and syringomycin, it contained Ser, Arg and "Dab". However,unlike the other pseudomycins, it did not have any Asx or Lys, but didhave the additional amino acids Gly, Ala, Pro, Tyr, Val and Leu. Theresults are summarized in Table 10.

Although not resolved by reverse-phase HPLC, Phe and Dab arebaseline-resolved by CE and can be quantified through theirUV-absorption. The "Dab" peaks from the amino acid analyzer were thuscollected and analyzed in this way. Electropherograms of "Dab" frompseudomycins A-D were essentially identical. Electropherograms frompseudomycin B and from syringomycin are presented in FIG. 10. The "Dab"peak from pseudomycin B was composed entirely of Dab, whereas the "Dab"peak from syringomycin, as expected, contained Dab and Phe in a molarratio of 2:1.

Pseudomycins A-C have amino acid compositions clearly different fromthat of syringomycin (Table 10). They have one fewer Ser and contain oneeach of Asx and Lys, residues which are not detected in syringomycin. Inaddition, these pseudomycins contain three residues of Dab, whereassyringomycin contains two of Dab and one of Phe.

Syringostatin A may be related to these pseudomycins but is structurallyunique, containing Ser, Thr, Dehydro-Thr, Dab, 3-hydroxyaspartic acid,chlorothreonine, homoserine, ornithine and a C₁₄ fatty acid.Syringotoxin, another pseudomonad antimycotic, has the same compositionas syringostatin A except that it contains one Dab residue and one Glyresidue. The composition of pseudomycin D is clearly different fromthose of any of the other compounds.

                  TABLE 10                                                        ______________________________________                                        Amino acid composition of pseudomycins                                        Molar percentage*                                                             Amino   Pseudo.  Pseudo.  Pseudo.                                                                              Pseudo.                                                                              Syringo-                              acid    A        B        C      D      mycin                                 ______________________________________                                        3(OH)Asp†                                                                      +        +        +      -      +                                     Asx     14-1 (1) 14-7 (1) 16-0 (1)                                                                             0-7 (0)                                                                              1-2 (0)                               Glx     2-1 (0)  2-9 (0)  1.1 (0)                                                                              0-9 (0)                                                                              0-6 (0)                               Ser     14-6 (1) 15-9 (1) 15-5 (1)                                                                             6-4 (2)                                                                              24-4 (2)                              Gly     3-1 (0)  1-9 (0)  1-4 (0)                                                                              3-6 (1)                                                                              0-8 (0)                               His     2-4 (0)  2-5 (0)  3-5 (0)                                                                              0-0 (0)                                                                              2-0 (0)                               Arg     10-6 (1) 10-2 (1) 12-0 (1)                                                                             3-8 (1)                                                                              13-4 (1)                              Thr     1-2 (0)  0-8 (0)  0-3 (0)                                                                              0-7 (0)                                                                              0-5 (0)                               Ala     1-2 (0)  1-6 (0)  0-7 (0)                                                                              36-3 (10)                                                                            1-3 (0)                               Pro     0-5 (0)  0-0 (0)  0-0 (0)                                                                              4-4 (1)                                                                              0-4 (0)                               Tyr     0-0 (0)  0-0 (0)  0-2 (0)                                                                              3-6 (1)                                                                              0-0 (0)                               Val     0-2 (0)  0-5 (0)  0-0 (0)                                                                              18-1 (5)                                                                             0-9 (0)                               Met     0-8 (0)  0-0 (0)  0-9 (0)                                                                              0-0 (0)                                                                              0-0 (0)                               Cys     0-0 (0)  0-0 (0)  0-0 (0)                                                                              0-7 (0)                                                                              0-0 (0)                               Ile     0-2 (0)  0-0 (0)  0-7 (0)                                                                              0-1 (0)                                                                              0-2 (0)                               Leu     0-0 (0)  0-0 (0)  0-0 (0)                                                                              5-1 (2)                                                                              0-6 (0)                               Dab     38-2 (3) 37-9 (3) 36-5 (3)                                                                             15-3 (5)                                                                             39-2 (3)                              Lys     11-0 (1) 11-1 (1) 11-1 (1)                                                                             0-3 (0)                                                                              0-2 (0)                               ______________________________________                                         *Samples were hydrolysed, derivatized and analysed as described in            Methods: molar percentages [(mol of each amino acid)/(mol of all amino        acids) × 100] are presented along with their estimated integer          values (in parentheses). Pseudomycins A-C were run in duplicate,              pseudomycin D in triplicate, and syringomycin as a single sample. The mea     total number of nanomoles of amino acids for each of these samples was 63     37, 22, 76 and 191, respectively.                                             †Separate analyses were done to identify 3hydroxyaspartic acid         [3(OH)Asp]. Plus and minus signs signify the presence or absence,             respectively, of this amino acid.                                        

Structural Comparison of the Pseudomonad Antimycotics

Amino acid modifications may account for the unique but similarchromatographic, electrophoretic and mass spectrometric behavior ofpseudomycins A-C. In syringomycin, for example, fatty acidesterification, chlorination, hydroxylation and/or dehydration of thepeptide have been reported. The 16 Da difference between pseudomycins Aand B could arise from esterifications with hydroxylated andunhydroxylated fatty acids, respectively. Similarly, pseudomycins A andC may contain fatty acids differing in length by two carbons (28 Da).The inability of composition analysis to identify all such structuraldifferences is not surprising since modified amino acids can be alteredor destroyed during acid hydrolysis or may produce PTC-amino acidderivatives which cannot be analyzed using the automated chemistrycurrently available.

Pseudomycins A-C share other characteristics of syringomycin andsyringostatin A; for example being cyclic esters and amino-terminallyN-acylated. Solvent and pH stability experiments have suggested this(Table 9), as have data from alkaline hydrolysis (Table 7). Additionalsupport comes from the fact that automated Edman degradations have notyielded any useful sequence information from these pseudomycins.Amino-terminal pyroglutaminylation does not seem to explain this failureof the Edman chemistry, using the criterion that pyroglutamageaminopeptidase treatment does not result in successful sequenceanalysis.

The theoretical average masses of syringomycin and pseudomycin A may becalculated. The lactone of syringomycin contains, in addition to thoseresidues quantified by amino acid analysis (Table 10), one each of3(OH)Asp, 4-chlor-threonine, Dab and 3-hydroxydodecanoic acid, yieldinga total mass of 1226.8 Da. If one postulates that pseudomycin A containsone each of 3(OH) Asp, 4-chlorothreonine and 3-hydroxydecanoic acid, inaddition to those residues reported in Table 10, its lactone would havea total mass of 1224.8 Da. These masses agree, within 0.2 and 0.1 Da,respectively, with those actually observed (Table 9).

Plant Material

The sources of the plant material used for testing phytotoxic activityof the antimycotic are listed in Table 11. All plants were grown undercontrolled conditions with 8 hrs. of darkness at 28° C. and 16 hrs. oflight at 32° C. per day.

                  TABLE 11                                                        ______________________________________                                        Sources of Plant Materials                                                    Common             Plant                                                      Name    Genera     Material   Source                                          ______________________________________                                        Corn    Zea        Seed       Burpee's                                        Rice    Oryza      Seed       Gary Strobel, MSU*                              Wheat   Triticum   Seed       MSU seed lab                                    Sugarcane                                                                             Saccharum  Whole Plant                                                                              Gary Strobel, MSU                               Timothy Phleum     Seed       Doug Kenfield, MSU                              Tall fescue                                                                           Festuca    Seed       Doug Kenfield, MSU                              Crabgrass                                                                             Digataria  Seed       Doug Kenfield, MSU                              Sunflower                                                                             Helianthus Seed       Burpee's                                        Tomato  Lycopersicon                                                                             Seed       Burpee's                                        Cucumber                                                                              Cucumis    Seed       Burpee's                                        Geranium                                                                              Geranium   Whole Plant                                                                              Ernst's Home Center                             Carob   Cerantonia Seed       Tunisia                                         Crown of                                                                              Euphorbia  Whole Plant                                                                              Gary Strobel, MSU                               Thorns                                                                        Sicklepod                                                                             Cassia     Seed       Doug Kenfield, MSU                              ______________________________________                                         *MSU-Montana State University                                            

Assay for Phytotoxic Activity

Individual leaves were placed in a sterile petri dish and kept at highhumidity by placing moist filter paper in the bottom of the dish. Thesolution to be assayed was dissolved in 0.05% TFA and applied to theleaf using a leaf puncture droplet overlay technique. An excised leafwas punctured using a Hamilton syringe, then a drop of the test samplewas applied to the puncture wound. When testing partially purifiedpseudomycin from the acetone precipitation procedure, 1, 10, and 100 μgwere applied. The control was 0.05% TFA and was applied in the samemanner. Each petri dish was sealed with parafilm and incubated for amaximum of 4 days at room temperature, under light.

Fungal Strain Source

The source of the fungal strains used to test antimycotic activity arelisted in Table 12.

                  TABLE 12                                                        ______________________________________                                        Fungal strain sources.                                                        Fungus          Source                                                        ______________________________________                                        Cephalosporium gramineum                                                                      Dr. Donald Mathre, MSU*, MT                                   Pyrenophora teres                                                                             Dr. Mike Bjarko, MSU, MT                                      Pyrenophora graminea                                                                          Dr. Mike Bjarko, MSU, MT                                      Rynchosporium secalis                                                                         Alfredo Martinez, MSU, MT                                     Ceratocystis ulmi                                                                             Dr. Gary Strobel, MSU                                         Rhizoctonia solani                                                                            ATCC 28268                                                    Botrytis alli   UCD 1159                                                      Sclerotinia sclerotiorum                                                                      Dr. David Sands, MSU                                          Verticillium dahliae                                                                          Dr. Donald Mathre, MSU, T-9                                   Verticillium dahliae                                                                          Dr. Donald Mathre, MSU, SS-4                                  Thielaviopsis basiola                                                                         Dr. Donald Mathre, MSU, CA                                    Fusarium oxysporum                                                                            ATCC E16322                                                   Fusarium culmorum                                                                             Bill Grey, MSU, MT                                            Fusarium graminearum                                                                          Bill Grey, MSU, MT                                            ______________________________________                                         *MSU-Montana State University                                            

Assay for Antimycotic Activity Against Plant Pathogenic Fungi

Partially purified pseudomycin (after Amberlite XAD-2 column) wasdissolved in 50% 1-propanol with 0.1% TFA at a concentration of 10μg/μl. The solution (10 μl) was spotted onto the appropriate solidmedium (Table 13) and allowed to dry. The solvent (10 μl) alone wasspotted adjacent to the pseudomycin as a negative control. Each platewas oversprayed with a sterile water suspension of the fungus, sealedwith parafilm, and incubated accordingly. Incubation time andtemperature varied according to the fungus to be tested (Table 13).

                  TABLE 13                                                        ______________________________________                                        Growth conditions used for plant pathogenic fungi                             in the assay for antimycotic activity.                                        Organism        Media      Temperature                                        ______________________________________                                        Rynchosporium secalis                                                                         Lima Bean  15°                                         Ceratocystis ulmi                                                                             PDA        23°                                         Cephalosporium sp.                                                                            PDA        23°                                         Pyrenophora teres                                                                             PDA-V8     15°                                         Pyrenophora graminea                                                                          PDA-V8     15°                                         Rhizoctonia solani                                                                            PDA        23°                                         Botrytis alli   PDA        23°                                         Sclerotinia sclerotiorum                                                                      modified CD                                                                              23°                                         Verticillium albo-atrum                                                                       PDA        23°                                         Verticillium dahliae                                                                          PDA        23°                                         Thielaviopsis basicola                                                                        PDA        23°                                         Fusarium oxysporum                                                                            Corn Meal  23°                                         Fusarium graminearum                                                                          Corn Meal  23°                                         Fusarium culmorum                                                                             Corn Meal  23°                                         ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Pseudomycin Antimycotic Activity Towards Plant                                Pathogenic Fungi                                                              Fungus              Activity                                                  ______________________________________                                        Rynchosporium secalis                                                                             +                                                         Ceratocystis ulmi   +                                                         Cephalosporium gramineum                                                                          -                                                         Pyrenophora teres   -                                                         Pyrenophora graminea                                                                              -                                                         Rhizoctonia solani  +                                                         Botrytis allii      -                                                         Sclerotinia sclerotiorum                                                                          +                                                         Verticillium albo-atrum                                                                           +                                                         Verticillium dahliae                                                                              +                                                         Thielaviopis basicola                                                                             +                                                         Fusarium oxysporum  +                                                         Fusarium graminearum                                                                              -                                                         Fusarium culmorum   +                                                         ______________________________________                                    

Assay for Antibacterial Activity

The same screen as described for the fungi was used to test theantimycotic sensitivity of three plant bacterial pathogens; 1)Corynebacterium michigenese, a gram positive organism, 2) Xanthomonascampestris, a gram negative organism, and 3) P. syringae MSU 16H, theorganism that produces the antimycotic. Pseudomycin (10 μl) was spottedonto Cory media for C. michigenense and King's B for X. campestris andsyringae, at concentrations of 250, 25, 2.5, 0.25, and 0.025 μg/μl. Theywere then oversprayed with ≈2×10¹¹ org./ml and incubated at roomtemperature for 48 hours. Both X. campestris and C. michigenense wereinhibited by a total of 2.5 mg and weakly inhibited by a total of 0.25mg. These are relatively high concentrations required for inhibition. P.syringae was not inhibited by the antimycotic.

Phytotoxic Activity

Partially purified pseudomycin (from Amberlite XAD-2 column) was used totest phytotoxicity in a total of six different monocots and sevendifferent dicots. One hundred, 10, and 1 μg of pseudomycin, dissolved in0.05% TFA was applied to a puncture wound on the leaf surface. Afterincubation for four days at room temperature in a moist petri dish, theleaves generally developed zones of necrosis surrounded by an area ofchlorosis. This is similar to the symptoms that develop with leafblight. In addition to the monocots displayed brown runners and thedicots venal necrosis. All of the monocots were affected by theantimycotic. Three of the dicots were not affected at theseconcentrations; carob (Cerantonia), cucumber (Cucumis), and geranium(Geranium) (Table 15).

                                      TABLE 15                                    __________________________________________________________________________    Phytotoxicity of partially purified pseudomycin. (N) = necrosis, (C) =        chlorosis, (V) = vein necrosis, (R) = brown runners, (W) = water              soaking,                                                                      (G) = green island. The response was estimated to be weak (+), moderate       (++),                                                                         or severe (+++). (-) = no difference relative to the control.                 Amount Applied (μg)     Amount Applied (μg)                             Monocots                                                                            100 10  1   Dicots   100 10  1                                          __________________________________________________________________________    Timothy                                                                             NCR NCR NCR Sunflower                                                                              NC  NC  NC                                               +++ +++ ++           ++  ++  +                                          Corn  NCW NCW NCW Carob    --  --  --                                               +++ +++ ++                                                              Tall Fescue                                                                         GCR GCR GCR Cucumber --  --  --                                               +++ +++ ++                                                              Crabgrass                                                                           WNC WNC WNC Tomato   NV  NV  --                                               +++ +++ +++          ++  ++                                             Sugarcane                                                                           NR  NR  NR  Geranium --  --  --                                               +++ +++ +                                                               Wheat NC  NC  NC  Crown of Thorns                                                                        NV  NV  NV                                               +++ +++ +            +++ ++  +                                                            Sicklepod                                                                              NV  NV  NV                                                                    +++ ++  +                                          __________________________________________________________________________

                  TABLE 16                                                        ______________________________________                                        Growth conditions used for plant pathogenic fungi                             in the assay for antimycotic activity.                                        Organism        Media      Temperature                                        ______________________________________                                        Rynchosporium secalis                                                                         Lima Bean  15°                                         Ceratocystis ulmi                                                                             PDA        23°                                         Cephalosporium sp.                                                                            PDA        23°                                         Pyrenophora teres                                                                             PDA-V8     15°                                         Pyrenophora graminea                                                                          PDA-V8     15°                                         Rhizoctonia solani                                                                            PDA        23°                                         Botrytis alli   PDA        23°                                         Sclerotinia sclerotiorum                                                                      modified CD                                                                              23°                                         Verticillium albo-atrum                                                                       PDA        23°                                         Verticillium dahliae                                                                          PDA        23°                                         Thielaviopsis basicola                                                                        PDA        23°                                         Fusarium oxysporum                                                                            Corn Meal  23°                                         Fusarium graminearum                                                                          Corn Meal  23°                                         Fusarium culmorum                                                                             Corn Meal  23°                                         ______________________________________                                    

Antifungal chemotherapy is limited both in the number of availableagents and in therapeutic applications. Many of the antifungal agentsare toxic even at low concentrations. For example, Amphotericin B, usedfor the treatment of superficial Candida infections, is active in vitroat concentrations of 0.01-2 μg/ml. It is also highly toxic, and someimpairment of renal function is seen in all patients regardless ofdosage given. New chemicals are needed for human antifungal agents.

One of the requirements of a human antifungal agent is stability inblood serum. Pseudomycin remains active in blood serum.

In order to test the stability of pseudomycin in human serum, humanwhole blood was allowed to clot, then centrifuged at 4000×G to separatethe serum from the cells. Partially purified pseudomycin was suspendedin the serum, in duplicate tubes, at concentrations of 50, 5, 0.5, 0.05,and 0.005 μg/μl. The antimycotic was dissolved in water, at the sameconcentrations, for the control. One set of tubes was incubated for twodays at room temperature and one set at 37° C. The antimycotic wastested for activity at 24 and 48 hours using Geotrichum as the assayorganism. The pseudomycin remained active in both the serum mixtures andthe control for the full 48 hours.

Furthermore, a partially purified preparation of pseudomycin is notendotoxic. This is especially important where the pseudomycin is usedfor intravenous treatment. In the situation wherein a human or animalpatient is afflicted with a deep systemic infection, injection of theantimycotic is typically necessary for treatment. Endotoxins generallyinclude the lipopolysacchrides from the cell walls of gram negativebacteria. When minute amounts of endotoxins are exposed to Linulusamoebocyte lysate, the lysate increases in viscosity and eventuallygels. This is the basis of the Etoxate test. Three different sampleswere tested for the presence of endotoxin; 1) BuOH crude extract, 2) TLCeluate and, 3) Amberlite XAD-2 eluate. All glassware was soaked in a 4%solution of dimethyldichlorosilane, then rinsed thoroughly with doubledistilled water. The glassware was then heat cured at 100° C. overnight.

Twelve of the silanized tubes were prepared as indicated in Table 17.Each sample was mixed with amoebocyte lysate alone (Table 18, tubes A)to test for the presence of endotoxin. The samples were also mixed withlysate and a known amount of endotoxin standard (tubes B). Thepseudomycin, water, and endotoxin standard dilutions were added first,followed by the E-toxate working solution. Each tube was mixed gently,then incubated for one hour undisturbed at 37° C. These tubes test forthe presence of an inhibitor in the sample.

                                      TABLE 17                                    __________________________________________________________________________    Contents of tubes for the endotoxin assay. All samples were dissolved         in endotoxin freewater and adjusted to pH 6.5 with endotoxin free NaOH.       Tubes                   Sample                                                A Tests for endotoxin in sample                                                                       1 BuOH crude extract                                  B Tests for etoxate inhibitor in sample                                                               2 eluted from TLC plate                               C Negative control      3 Amberlite XAD-2 column                              D-H Standard dillutions                                                       __________________________________________________________________________    Tubes                                                                         Contents                                                                            A1 A2 A3 B1  B2  B3  C  D   E   F   G   H                               __________________________________________________________________________    Sample                                                                              .1 ml                                                                            .1 ml                                                                            .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                  Water                      .1 ml                                              Endotoxin      .004 μg                                                                        .004 μg                                                                        .004 μg                                                                           .008 μg                                                                        .004 μg                                                                        .002 μg                                                                        .001 μg                                                                        .0005 μg                     Standard                                                                      Etoxate                                                                             .1 ml                                                                            .1 ml                                                                            .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                           __________________________________________________________________________

                                      TABLE 18                                    __________________________________________________________________________    Results of Etoxate test for endotosins. All samples were dissolved            in endotoxin freewater and adjusted to pH 6.5 with endotoxin in free          NaOH.                                                                         Tubes                    Sample                                               A Tests for endotoxin in sample                                                                        1 BuOH crude extract                                 B Tests for etoxate inhibitor in sample                                                                2 eluted from TLC plate                              C Negative control       3 Amberlite XAD-2 column                             D-H Standard dillutions                                                       __________________________________________________________________________    Tubes                                                                         Contents                                                                            A1 A2 A3 B1  B2  B3  C  D   E   F   G   H                               __________________________________________________________________________    Sample                                                                              .1 ml                                                                            .1 ml                                                                            .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                  Water                      .1 ml                                              Endotoxin      .004 μg                                                                        .004 μg                                                                        .004 μg                                                                           .008 μg                                                                        .004 μg                                                                        .002 μg                                                                        .001 μg                                                                        .0005 μg                     Standard                                                                      Etoxate                                                                             .1 ml                                                                            .1 ml                                                                            .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                                                                            .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                                                                             .1 ml                           Results                                                                             -  -  -  -   +   -   -  +   +   +   +   +                               __________________________________________________________________________

After incubation, the tubes were removed, one at a time, and slowlyinverted 180° while observing for evidence of gelation. A positive testis the formation of a hard gel which permits complete inversion of thetube or vial without disruption of the gel. All other results wereconsidered negative.

The BuOH crude extract and the eluate from the XAD-2 Amberlite columntested negative for endotoxin (tube A), but the corresponding tube B wasnegative. This means there is an inhibitor in the sample and the test isinvalid. The sample from TLC tested negative for endotoxin and theinhibitor. This test indicates a certain level of purity required forapplied use of this toxin. The minimum inhibitory concentration of thetoxin against seven human fungal pathogens was estimated using a serialtube dilution technique. The toxin was most effective at inhibiting theyeast fungi, Candida and Cryptococcus (Table 19).

                  TABLE 19                                                        ______________________________________                                        Minimal inhibitory concentrations of                                          pseudomycin A against fungal pathogens of humans.                                           MIC (μg ml.sup.-1)*                                          Organism        24 h      48 h   72 h                                         ______________________________________                                        Candida albicans 89-80                                                                        3-12      25     ND                                           Candida albicans 89-96                                                                        3-12      12-5   ND                                           Candida tropicalis                                                                            3-12      6-25   ND                                           Cryptococcus neoformans                                                                       1-56       3-1   ND                                           Bipolaris spicifera                                                                           ND        12-5   >50                                          Aspergillus fumigatus                                                                         ND        12-5     50                                         ______________________________________                                         *MIC is defined as the minimal concentration required to cause an             inhibition of fungal growth as judged by the turbidity of the broth           suspension.                                                              

In order to test the minimum inhibitory concentrations for human fungalpathogens, the following was performed.

A serial dilution tube test was used in which the toxin was diluted outin tubes containing equal amounts of the fungus to be tested which weresuspended in the appropriate medium. The tubes were incubated at roomtemperature or 30° C. for the mould fungi or yeast fungi, respectively.After incubation, the last tube in the dilution series which inhibitedthe growth of the fungi, was recorded as the minimum inhibitoryconcentration.

The following describes the methods and further characterization of thepseudomycins of the present invention.

Microbiological Methods

The P. syringae MSU 16H strain is an elm tree acclimated transposon (fn903) generated mutant of the wild type strain MSU 174. It is equivalentto MSU strain 206 previously described in (Harrison et al., (1991), J.Gen. Microbiol. 137, 2857-2865). It was grown in still culture underconditions reported in (Ballio et al. (1990) FEBS Lett. 269; 377-380).Antifungal activity was assayed on Rhodotorula pilmanae (Sinder et al.(1991) Physiol. Plant Pathol. 1, 199-213).

Preparation of Pseudomycins

After 9-10 days growth at 25° C. the bioactive metabolites wereextracted and partially purified according to Bidwai et al. (Bidwai etal. (1987) Plant Physiol. 83: 39-43), and finally fractionated byreverse phase HPLC on an Aquaspore RP300 column (4.6×250 mm, 7 μm ID.Applied Biosystems) using a Beckman System Gold 126 instrument underconditions described in (Iacohellis et al. (1992) Physiol. Mol. PlantPathol. 40:107-116). Individual peaks were freeze-dried, quantitated byamino acid analysis after HCl hydrolysis, and assayed for activitytoward R. pilimanae.

Analytical Methods

Amino acids were analyzed out-as reported in (8) except that anEppendorf-Biotronik LC 3000 analyzer was used. Some analyses were alsoperformed by GC-MS after transformation into TBDMS derivatives (ChavesDas Neves et al. A.M.P. (1987) J. Chromatogr, 392, 249-258). Thechirality of amino acid residues was determined by Marfey's method(Marfey, P. (1984) Carlsberg Res. Comm. 591-596). Peptide sequences wereobtained by automated Edman degradation using an Applied Biosystemsmodel 476A sequencer. Samples were spotted on Problott membrane (AppliedBiosystems) and sequenced with a Blott Cartridge (Applied Biosystems).

Table 20 shows an amino acid comparison (integral values) for thepseudomycins using syringomycin as a comparison.

                                      TABLE 20                                    __________________________________________________________________________    Amino acid (integral values) for the pseudomycins using syringomycin as       comparison*                                                                              Molar Percentage                                                   Amino acid Pseudomycin A                                                                         Pseyudomycin B                                                                         Pseudomycin C                                                                         Syringomycin                              __________________________________________________________________________    Lysine     11.0 (1)                                                                              11.1 (1) 11.1 (1)                                                                              0.2 (0)                                   Aspartic acid                                                                            14.1 (1)                                                                              14.7 (1) 16.0 (1)                                                                              1.2 (0)                                   Serine     14.6 (1)                                                                              15.9 (1) 15.5 (1)                                                                              24.4 (2)                                  "arginine" 10.6 (1)                                                                              10.2 (1) 12.2 (1)                                                                              13.4 (1)                                  (chlorothreonine)**                                                           diaminobutyric acid                                                                      38.2 (3)                                                                              37.9 (3) 36.5 (3)                                                                              39.2 (3)                                  phenylalanine and                                                             2,3 dehydro 2 amino                                                           butyric acid                                                                  hydroxy aspartic                                                                         + (1)   + (1)    + (1)   + (1)                                     acid***                                                                       TOTAL RESIDUES                                                                           8       8        8       7                                         __________________________________________________________________________     *Each sample was hydrolyzed, derivatized and analyzed according to            standard methods. The numbers in each column show the exact molar             percentages of each amino acid that was recovered for each hydrolyzed         sample.                                                                       **Chlorothreonine seems to have the same retention time as arginine           **Data impossible to obtain without the presence of a standard for            comparison, an estimated residue amount is given.                             NOTE:                                                                         Threonine, itself was not found by this analysis. It may have been            destroyed by acid hydrolysis. It is an integral amino acid in the             structure (see FIG. 1).                                                  

Given that there are technical problems in obtaining correct analyticaldata by amino acid analysis, the inventors set out to make a completestructural determination of pseudomycins A, B and C. They now used new,more highly sensitive techniques, including high resonance NMR. The dataobtained by this, and other methods described below have provided thechemical structure of pseudomycins A, B and C. The new data, supplantearlier reported analytical methods because of their sensitivity andsuperiority. These methods have allowed for a determination of thecomplete structure of pseudomycins A, B and C.

FAB-MS spectra were recorded on a VG ZAB 2 SF instrument equipped with acesium gun operating at 25 kV 2 pA. Samples dissolved in 5% acetic acidwere directly loaded onto the probe tip coated withglycerol/thioglycerol 1:1.

NMR spectra were run at 27° C. on a Bruker AMX600 instrument operatingat 600.13 MHz. Samples (1 mg) were dissolved in 0.8 ml of either D₂ O orH₂ O/D₂ O 90:10, at pH 4.8. 2D NMR experiments were performed in thephase-sensitive mode with TPPI phase cycle (Marion, D. et al. (1983)Biochem. Biophys. Res. Comm. 113, 967,974) typically using 2K of memoryfor 512 increments. The number of scans were optimized in order toobtain a satisfactory signal-to-noise-ratio. Total Correlationexperiments (TOCSY) were performed using the MLEV-17 spinlock compositepulse sequence (Braunschweiler et al. (1983) J. Magn. Reson, 53,521-528; and Bax. A. et al. (1985) J. Magn. Reson. 65, 355-360) with atypical mixing time ranging from 60 to 120 ms (relayed) in order toobserve either direct or remote connectivities. NOE dipolar correlated2D spectra were obtained using the NOESY pulse sequence (Wuthrich et al.(1984) J. Magn. Reson 58, 370-388).

The mixing time for the magnetization exchanges ranged from 60 to 220ms. Data were processed on a microVax II graphics workstation by the"TRITON" 2D NMR software of R. Boelsen and G. Vuister, kindly providedby prof. R. Kaptein of Utrecht University. FIDS were weighted by asinebell apodization function shifted typically π/3 in both dimensions.In all homonuclear 2D experiments, a matrix 1024×1024 in thephase-sensitive mode was thus obtained with a digital resolution of ≈5Hz/point. A baseline correction was carried out in both dimensions usinga polynomial fit. ¹³ C-¹ H heteronuclear correlations were obtained inthe reverse-detection mode on the AMX600 BRUKER instrument (1K×512 w).

Chemical Methods

The lactone ring hydrolysis was obtained by 3 h incubation at 37° C.with 50 mM ammonium bicarbonate.

Enzymatic Hydrolyses

Lipodepsipeptides (250 μg) dissolved in 0.1M ammonium bicarbonate (15μl) pH 8.0 were incubated at 37° C. for >h with TPCK trypsin(Worthington Biochemicals Co.) using an enzyme/substrate ratio of 1:30.After lyophilization the hydrolysis products were fractionated by HPLCas described under 2.2. Elution was performed with a solvent gradientobtained by mixing 0.2% TFA in water with 70% acetonitrile containing0.2% TFA, flow rate 0.8 ml/min. The main peaks were freeze-dried and thesamples were analyzed by FAB-MS and Edman degradation.

Reverse phase HPLC of a P. syringae MSU 16H extract partially purifiedaccording to Bidwai et al. (Bidwai et al. (1987) Plant Phsiol. 83:39-43) produced an elution pattern of the same type observed withsyringomycin and syringotoxin-producing strains (FIG. 11) (Ballio et al.(1991) FEBS Lett. 291: 109-112). Several peaks appeared in the regionwhere the lipodepsinonapeptides are eluted, followed by two morehydrophobic peaks emerging from the column at higheracetonitrile-isopropanol concentration. FAB-MS and amino acid analyses(see below) of the substances isolated from the six more relevant peaksindicated that four corresponded to the previously described (Harrisonet al., (1991), J. Gen. Microbiol. 137, 2857-2865) pseudomycin A, B, Cand D, another (C') could be a further pseudomycin. (Harrison et al.,(1991), J. Gen. Microbiol. 137, 2857-2865).

The amino acid composition (see below) clearly indicated that thesubstances isolated from peaks A, B, C and C' were different from knownP. syringae metabolites. The identify of the two compounds from peaks Dand D' with the two syringopeptins was proved by the same MH⁺ values,absorbance at 280 nm. HPLC elution times and detailed ¹ H-NMR data.

The complete structure of compounds contained in peaks A, B, C and C'was elucidated by the use of 2D NMR, FAB-MS and chemical and enzymaticdegradations carried out on microquantities. Pseudomycin A, a relativelyabundant component, was at first investigated. Amino acid determination,both by conventional ion-exchange chromatography and by GC-MS afterderivatization with N-methyl-N-TBDMS-trifluoroacetamide (Chaves DasNeves et al. A.M.P. (1987) J. Chromatogr., 392, 249-258) showed thepresence of one mole each of Ser, aThr, Asp, Asp(3-OH), Thr(4-Cl), Lys,and two moles of Dab.

The methods commonly used for 2D NMR studies reached the same conclusionand furthermore identified a Z-Dhb residue and the3,4-dihydroxytetradecanoyl moiety; the chemical shifts and assignmentsof ¹ H and ¹³ C-NMR spectra are reported in Table 21. All amino acidresidues, with the exception of one Dab, had the L configuration.

                                      TABLE 21                                    __________________________________________________________________________    Structure of pseudomycins, syringoslatins, syringotoxin and                   syringomycins.                                                                Me(CH.sub.2).sub.n --CH(X)--CH(OH)--CH.sub.2 --CO--L--Ser-aa.sub.2            -aa.sub.3 -aa.sub.4 -aa.sub.5 -aa.sub.6 -Z--Dhb--L--Asp(3-OH)--L--Thr(4-Cl            n     X      aa.sub.2                                                                             aa.sub.3                                                                             aa.sub.4                                                                             aa.sub.5                                                                             aa.sub.6                     __________________________________________________________________________    PSs A, C                                                                              9,11  OH                                                                                   D--Dab L--Asp L--Lys L--Dab L--αThr                PSs B, C.sup.1                                                                        9,11  H                                                               SSs     7,9   H, OH  D--Dab L--Dab D--Hse L--Orn L--αThr                ST      9     H      D--Dab Gly    D--Hse L--Orn L--αThr                SRs     5,7,9 H      D--Ser D--Dab L--Dab L--Arg L--Phe                       __________________________________________________________________________

The presence in the molecule of nine amino acid residues and a longchain hydroxyacyl, the difference of 18 mass units between thecalculated sum of the residues and the molecular weight found by FAB-MS(MH⁺ 1223-1225: the doublet indicates the presence of one chlorineatom), the observed addition of one mole of water (MH⁺ 1241-1243) bytreatment with ammonium bicarbonate (followed by substitution ofchlorine with OH:MH⁺ 1223 singlet), FAR-MS of a sample treated withammonium bicarbonate produced a fragmentation pattern in agreement withthe following partial sequence:Asp-Lys-Dab-aThr-Dhb-Asp(3-OH)-Thr(4-OH), where Thr(4-OH) arises fromThr(4-Cl) at basic pH. The occurrence of an L-lysine residue prompted tocleave pseudomycin A by trypsin.

After incubation with the enzyme the solution was fractionated byreverse phase HPLC. The fragment present in the main hydrophilic peakcorresponded to the C-terminal part of the molecule, as ascertained byautomated Edman degradation which yielded a Dab residue on the firstcycle and a Thr residue on the second; the sequence determination wasstopped in correspondence of the dehydro-amino acid (Wakamiya et al.(1985) Tetrahedron Lett. 26, 665-668).

Treatment of the same fragment by a modified Marfey's procedure (Marfey,P. (1984) Carlsberg Res. Comm. 591-596) allowed to assign theL-configuration to the Dab residue adjacent to Lys and consequently theD-configuration to the other Dab residue. The FAB-MS spectrum of aprominent more hydrophobic peak showed a pseudomolecular ion (MH⁺ 691)expected for the rest of the molecule, namely for3,4-dihydroxytetradecanoyl-(Dab,Ser)-Asp-Lys-OH.

The complete sequence of the tetrapeptide moiety and the site ofacylation emerged from NMR spectra of pseudomycin A (see Table 21). Thecomplete structure of pseudomycin A is reported in FIG. 13, where thearrows indicate the short range strong NOE contacts which have allowedto elucidate, independently from the chemical approach, the amino acidsequence, as well as the position and the type of closure of the macroring.

A number of long range NOE contacts have also been identified; theseproximities, together with available information about the chirality ofthe amino acid residues, are prerequisites for the determination of thesolution structure of this molecule.

Pseudomycins B(MH¹ 1207-1209), C (MH⁺ 1251-1253) and C'(MH⁺ 1235-1237)are closely related to pseudomycin A. In fact, amino acid composition,and fragmentation observed in the FAB-MS spectra after lactone openingwith ammonium bicarbonate gave identical results for all fourpseudomycins. Thus, very likely they differ only for the long chain acylgroup: their MH⁺ values suggest that the nonapeptide moiety is acylatedin pseudomycin B by 3-monohydroxytetradecanoate, a pseudomycin C by3,4-dihydroxyhexadecanoate and in pseudomycin C' by3-monohydroxyhexadecanoate. The position of the four pseudomycins in thereverse phase HPLC elution pattern is consistent with an increasedhydrophobicity on passing from A to C'.

As compared to the known lipodepsinonapeptides from P. syringae pv.syringae (Table 21), the pseudomycins have: a) the same variety of fattyacids (3-hydroxy and 3,4-dihydroxy) previously found in syringostatins,except that some have a longer aliphatic chain (C₁₆); b) an identicalN-terminal residue (L-Ser), N-acylated by the fatty acid and O-acylatedby the terminal carboxyl group; c) the same C-terminal tripeptide withthe carboxy group closing the lactone ring; d) a D-amino acid residue inthe second position, similarly to all so far described congenersobtained for isolates of P. syringae pv. syringae (Scaloni et al. (1994)Nat. Product Lett. (in press)); e) the third and the fourth residueswith the L-configuration, while in their congeners either one or theother has the D-configuration; f) the fifth residue correspondent to abasic L-amino acid residue (Dab), as found in syringomycins (Arg)(3,4)syringotoxin (Orn) (Ballio et al. (1990) FEBS Lett. 269; 377-380; andFukuchi et al. (1990) Agric. Biol. Chem. 54; 3377-3379) syringostatins(Orn)(Isogai et al. (1990) Tetrahedron Lett. 31, 695-698).

The occurrence of L-Asp in the peptide moiety of the pseudomycins is anovel feature of the lipodepsinonapeptides which appears to affect theirbiological properties.

In sum, the invention provides for a composition for the treatment offungal or bacterial infections comprising a substantially pure peptidepseudomycin derived from P. syringae MSU 16H and a pharmaceuticalcarrier, wherein the peptide comprises at least one peptide selectedfrom the group consisting of proteins Pseudomycin A, Pseudomycin B andPseudomycin C, each having a molecular weight (M+H⁺) of about 1224, 1208and 1252 Da, respectively and mixtures of said proteins. (Actual molweight 1223, 1207 and 1251 Da.) The composition may comprise one, two,three or all four peptides of the invention in any combination,including mixtures. Pseudomycins A and Pseudomycin C are particularlypreferred. The peptide pseudomycin of the invention may also besynthetically produced.

Pseudomycin A, Pseudomycin B, and Pseudomycin C comprises1-L-chlorothreonine, 1-L-serine, 1-D diaminobutyric acid, 1-L-asparticacid, 1-L-lysine, 1-L-diaminobutyric acid, 1-L-threonine, 1-2,3-dehydro2-aminobutyric acid and 1 L-3-hydroxy aspartic acid.

Many fungi cause opportunistic infections in immunologically compromisedpatients, such as those suffering from AIDS. The most common treatmentcurrently available for these systemic mycoses is administration of thehighly toxic antibiotic amphotericin B. Since pseudomycins retain theirantimycotic activity in human serum and are non-toxic in mice, they maybe used as an alternative to amphotericin B in the treatment of thesediseases.

Formulations of the pseudomycin protein of the invention may includefrom 0.01% to 99% pseudomycin protein and from 99% to 0.01% inertpharmaceutically acceptable carrier. Such carriers are known asevidenced by Remington's Pharmaceutical Sciences, 18th ed. (1992) MackPublishing Co., incorporated herein by reference in its entirety. Thepseudomycins may be administered by any methods known in the art asevidenced by Remington's Pharmaceutical Sciences. Dosages may also becalculated by methods known in the art. As is known in the art, dosagesare dependant upon the type of administration, with different dosagesfor topical, intravenus, intramuscular and oral administration.

The purpose of the above description and examples is to illustrate someembodiments of the present invention without implying any limitation. Itwill be apparent to those of skill in the art that various modificationsand variations may be made to the composition and method of the presentinvention without departing from the spirit or scope of the invention.All patents and publications cited herein are incorporated by referencein their entireties.

We claim:
 1. A composition comprising one or more isolated andsubstantially pure pseudomycin peptides obtained from P. syringae, saidone or more peptides being sufficiently pure and active to beadministered to plants or animals as an antibiotic without substantialside effects, wherein said one or more peptides are selected from thegroup consisting of:a) Pseudomycin A, having a molecular weight of about1223 Daltons; b) Pseudomycin B, having a molecular weight of about 1207Daltons; and c) Pseudomycin C, having a molecular weight of about 1251Daltons; wherein each of said Pseudomycins A, B and C are cyclic esters,and wherein each of said Pseudomycins A, B and C comprise the amino acidsequence ##STR1## wherein AA¹ =L-3-hydroxy-aspartyl AA²=L-4-chlorothreonyl Ser=L-serinyl Dab=D-diaminobutyryl ASP=L-aspartylandthe carboxyl group of AA² is joined to the serinyl hydroxyl group by anester linkage.
 2. The composition according to claim 1, wherein each ofsaid Pseudomycin A, B and C contains a long chain fatty acid.
 3. Thecomposition according to claim 2, wherein each of said Pseudomycin A, Band C contains the following amino acids in an equimolar ratio:L-chlorothreonine; L-serine; D-diaminobutyric acid; L-aspartic acid;L-lysine; L-diaminobutyric acid; L-threonine; 2,3-dehydro-2-aminobutyricacid; and, L-3-hydroxy aspartic acid.
 4. The composition according toclaim 1, wherein said composition comprises Pseudomycin A.
 5. Thecomposition according to claim 1, wherein said composition comprisesPseudomycin B.
 6. The composition according to claim 1, wherein saidcomposition comprises Pseudomycin C.
 7. The composition according toclaim 1, wherein said composition comprises Pseudomycin A andPseudomycin B.
 8. The composition according to claim 1, wherein saidcomposition comprises Pseudomycin A and Pseudomycin C.
 9. Thecomposition according to claim 1, wherein said composition comprisesPseudomycin B and Pseudomycin C.
 10. The composition according to claim1, wherein said composition comprises Pseudomycin A, Pseudomycin B andpseudomycin C.
 11. A pseudomycin peptide having the following formula:##STR2## wherein n is 9 or 11, and wherein R is --H or --OH.
 12. Thepseudomycin peptide according to claim 11, wherein n is 9, and wherein Ris --OH.
 13. The pseudomycin peptide according to claim 11, wherein n is9, and wherein R is --H.
 14. The pseudomycin peptide according to claim11, wherein n is 11, and wherein R is --OH.
 15. The pseudomycin peptideaccording to claim 11, wherein n is 11, and wherein R is --H.
 16. Thecomposition of claim 1 produced from an isolated, substantially purePseudomonas culture, wherein said Pseudomonas culture produces apseudomycin having substantially all of the antifungal characteristicsof a pseudomycin derived from P. syringae MSU 16H (ATCC 67028).
 17. Thecomposition of claim 16 produced from P. syringae MSU 16H (ATCC 67028).18. The composition of claim 1 produced from an isolated, substantiallypure Pseudomonas culture, wherein said Pseudomonas culture produces apseudomycin having substantially all of the antibacterialcharacteristics of a pseudomycin derived from P. syringae MSU 16H (ATCC67028).
 19. The composition of claim 18 produced from P. syringae MSU16H (ATCC 67028).
 20. A composition for the treatment of fungal orbacterial infections comprising an effective antifungal or antibacterialamount of one or more substantially pure pseudomycin peptides accordingto claim 11 and a pharmaceutically acceptable carrier.
 21. Thecomposition according to claim 20, wherein said composition comprises atleast two pseudomycin peptides.
 22. The composition according to claim20, wherein said one or more peptides are active in blood serum andsubstantially non-endotoxic.
 23. The composition according to claim 20,wherein the pharmaceutically acceptable carrier is selected from thegroup consisting of a cream, a lotion and an intravenous solution.
 24. Amethod of treating fungal or bacterial infections comprisingadministering an effective antifungal or antibacterial amount of acomposition according to claim 20 to a plant or animal afflicted with orsusceptible to fungal or bacterial infection.
 25. The method accordingto claim 24, wherein the infection comprises a fungal infection, andwherein the fungal pathogen is selected from the group consisting ofcandida abicans, candida tropicalis, cryptococcus neoformans, biolarisspecifera and aspergillus fumigatus.
 26. The method according to claim24, wherein the composition is administered directly to a surfaceinfection in the form of a cream or lotion or is administeredintravenously.
 27. The method according to claim 24, wherein saidcomposition is active in blood serum and is non-endotoxic.
 28. Themethod according to claim 24, wherein said composition is administeredto an immunologically compromised animal.